The present invention relates to a rapid, effective, economical and sensitive method for the detection of antibodies induced by a heparin substance and the diagnosis of immune or autoimmune pathologies potentiated by a heparin substance, such as heparin-induced thrombocytopenia (HIT type II). The invention also relates to a kit for the detection of heparin-dependent antibodies and the diagnosis of pathologies potentiated by a heparin substance, such as heparin-induced thrombocytopenia.
Thrombopenias or thrombocytopenias may be of many origins. They may be produced by the presence of drugs, notably quinine/quinidine, pentosan polysulfate, but above all heparin. (See article by M. C. Berndt et al., Blood Reviews 1, pages 111-118 (1987) and article by B. Tardy-Poncet, Am J Hematol 2994; 45(3): 252-7).
Heparin is administered to patients as an anti-coagulant factor for preventing risks of venous or arterial thromboses. However, certain patients treated with heparin develop antibodies induced by heparin, resulting in thrombocytopenia, which may be very severe or even fatal. These thrombocytopenias appear to be produced by IgG, IgM or IgA antibodies, which develop after 5 or more days of treatment with heparin. Antibodies of isotype IgG are by far the most pathogenic.
This reaction takes place at a “critical concentration” of heparin (Y. Gruel, Presse Med 1998; 27 (Suppl. 2): 7-12 and T E Warkentin et al., Chest 2004; 126: 311S-37S).
Rapid identification of patients at risk of or developing this HIT type II pathology appears to be essential.
At the present time, the methods for diagnosis of thrombocytopenia are:                blood platelet count before, during and after treatment, a long and not very specific method;        search for the absence of an etiology other than a thrombocytopenia (infection, other therapies, etc.), a long and tedious method;        use of biological tests seeking the presence of antibodies directed against platelets in the presence of the inducer drug;        search for and measurement by ELISA method or some other immunologic method for antibodies directed against heparin and platelet factor 4 (pF4) complexes.        
Of these biological tests, those principally used are platelet aggregation tests, which require suitable apparatus and the operational details of which are long and lack sensitivity.
Other methods that have been described [articles by J. G. Kelton et al., Blood 72 (No. 2), pages 925-930 (1988) and B. H. Chong, British Journal of Haematology 49, pages 531-540 (1988) and B. H. Chong, Blood Reviews 2, pages 108-114 (1986) and D. Sheridan et al., Blood 67 (No. 1), pages 27-30 (1986) and Y. Gruel, Sang Thrombose Vaisseaux, 1 (No. 4), pages 233-236 (1989)] employ study of platelet fixation of serum IgG, release of 14C-radiolabeled serotonin at two different concentrations of heparin, availability of platelet factor 4, fixation of the complement, inhibition of lysis of the complement and agglutination of sensitized red blood cells. These methods have the disadvantages of not being very sensitive or taking a long time to carry out.
Other methods that are sensitive and rapid in their performance, based on cytochemistry, use either marking of platelets activated or not, detected by flux cytometry, or purified factors issuing from platelets capable of preferentially binding to heparin and forming a complex with heparin-induced antibodies, or binding competition tests. All of these tests are performed with fresh platelets. Lastly, other immuno-cytochemical tests are done with heparin, plasma to be tested potentially containing heparin-induced antibodies and an antibody coupled to a reference molecule, but these last tests are performed without addition of platelets nevertheless necessary for sensitization of the test.
The determination of heparin-induced antibodies is essential in the prevention or treatment of HIT type II.
Several types of heparins are used:                non-fractionated heparins (NFH), which induce the appearance of heparin-induced antibodies sooner and more frequently;        heparins of low molecular weight.        
However, it is possible to use any type of heparin derivatives or of negatively charged non-glycosaminoglycan linear polymers that are not carbohydrates, such as polyvinyl sulfate, polyvinyl sulfonate, polystyrene sulfonate, polyanethol sulfonate, polyvinyl phosphates and polyvinyl phosphonate, poly-D glutamate (see PCT/US97/02840 WO9732211).
It has been discovered that the use of factors having a high affinity for heparin are very promising because they permit biological tests to be more specific and sensitive. In effect, heparin-induced antibodies have long been considered to be directed against heparin itself, but since the antigenic target of the antibodies has been identified, notably as described in EP 0,495,971, as being stoichiometic complexes of heparin and of platelet factor, pF4, more and more information concerning the mechanisms involved is available. In effect, pF4, in binding to heparin so as to neutralize its anticoagulant properties, changes conformation and then becomes immunogenic.
In this method, the factors issuing from platelets having a high affinity for heparin or the antibody inducer drug and obtained by platelet cleavage or lysis are:                platelet factor 4 or pF4        fractions of this factor pF4        fractions containing at least one substance washed out at the same time as pF4        recombinant pF4 and its variants        synthetic peptides absorbing all or part of the amino acid sequence of pF4        proteoglycan        proteoglycan-pF4 complexes        and mixtures thereof.        
However, such a method necessarily requires purification of platelet factor 4 (pF4) or the use of purified and functional recombinant pF4, which makes the method more complex and very burdensome.
U.S. Pat. No. 5,972,717 describes such a method for the detection of heparin-induced antibodies using a factor with high affinity for heparin adsorbed on a support, purified platelet factor pF4 and the serum or plasma of a patient to be tested. In this method, the factor with high affinity for heparin used is streptavidin, and it is used for the purpose of fixing the heparin molecule securely but especially for orienting it on the support. Further, use of the method as described requires the purification of pF4, a major disadvantage in terms of complexity and reliability as well as cost.
Another method for the detection of heparin-induced antibodies is described in the document entitled [in English:] “Antibodies to macromolecular platelet factor 4-heparin complexes in heparin-induced thrombocytopenia: a study of 44 cases,” [end English] published in Thrombosis and Haemostasis, Vol. 73, No. 1, 1995, pages 21 to 28. For detecting antibodies binding to the pF4-heparin complex, this document describes an ELISA assay using a substance (MBSS) coating the wells of the plate, a pF4-heparin mixture, a goat serum and the plasma to be tested. Although such a method may be promising, it nevertheless has some limits, principally as concerns its cost and its detection. In effect, this ELISA assay requires an essential purification of the pF4 antigen, a disadvantage already pointed out above. Moreover, the method as described only permits detection of pF4-dependent antibodies, whereas in the diagnosis of immune or autoimmune heparin-induced pathologies, it would be particularly interesting and advantageous to be able to detect all heparin-induced antibodies, i.e., all antibodies induced by antigen-heparin complexes, the antigen not being limited to only pF4.
The majority of recent methods of the prior art relating to this detection of heparin-induced antibodies use fixing of said heparin on a support and addition of an antigenic substance, such as, for example, platelet factor pF4, glycosaminoglycan or proteoglycan polymers, permitting formation of the complex generating detectable heparin-dependent antibodies. Nevertheless, all the documents of the prior art (U.S. Pat. No. 5,972,717, U.S. Pat. No. 5,753,445, U.S. Pat. No. 5,972,718, U.S. Pat. No. 5,466,582, the article entitled “Antibodies to macromolecular platelet factor 4-heparin complexes in heparin-induced thrombocytopenia: a study of 44 cases,” published in Thrombosis and Haemostasis, Vol. 73, No. 1, 1995, pages 21 to 28, the article entitled [in English:] “Polyarginine as a multifunctional fusion tag,” published in Protein Science: A publication of the Protein Society, June 2005, Vol. 14, No. 6, June 2005 (2005-06), pages 1538-1544) require at least one step of purification of the material used, which considerably increases the cost of the method. Thus, in the light of the prior art set forth above, it was necessary to perfect a method for the detection of heparin-induced antibodies at reduced cost and with increased sensitivity.